CN110643511A - Micro-fluidic chip and design method and application thereof - Google Patents

Micro-fluidic chip and design method and application thereof Download PDF

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CN110643511A
CN110643511A CN201910858617.XA CN201910858617A CN110643511A CN 110643511 A CN110643511 A CN 110643511A CN 201910858617 A CN201910858617 A CN 201910858617A CN 110643511 A CN110643511 A CN 110643511A
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channel
main channel
cells
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narrow
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林雪霞
宿建龙
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Huaqiao University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials

Abstract

The invention discloses a micro-fluidic chip and a design method and application thereof. The microfluidic chip comprises a main channel, a capture unit and a narrow channel; the main channel is used for transmitting cells and cell culture media, a plurality of capturing units are oppositely arranged on the side wall of the main channel at intervals and used for capturing and culturing single cells; the narrow channel is used for communicating the two capture units, forming an exchange channel between the captured single cells and preventing the captured single cells from flowing into the opposite main channel. The invention has simple manufacture and easy operation, can realize the capture and culture of two single cells, and carry out the research of the interaction between cells, and is suitable for researching the heterogeneity of the cells and the pathogenesis of diseases. The method can be used for developing the research of the tumorigenesis development mechanism, providing basis and new research thinking for constructing an in vitro effective tumorigenesis model, and providing certain theoretical support for tumorigenesis development.

Description

Micro-fluidic chip and design method and application thereof
Technical Field
The invention relates to a micro-fluidic chip and a design method and application thereof, which are used for capturing and culturing single cells in two cells and researching the interaction between the cells.
Background
A single cell is an essential unit constituting a living body, and conventional cell research is always performed on a group of cells. The obtained result is often an average value of a group of cells, information difference between individuals is covered, and in proteomics research, behaviors and reactions of cells in different environments are completely different when the same cell is in different periods, and the heterogeneity characteristics of the cells are most obvious in tumor cells and stem cells. The micro total analysis system, also called microfluidic chip or lab-on-a-chip (LOC), is a technical platform for replacing various functions of conventional chemical or biological laboratories by integrating or basically integrating basic operation units related to sample preparation, reaction, separation, detection, cell culture, sorting, lysis, etc. on a chip of several square centimeters (even smaller) and forming a network by microchannels, so that a controllable fluid can penetrate through the system. The one-key closed operation of capturing, identifying and detecting from the single cell can be realized by utilizing the microfluidic chip technology, the efficiency is high, and the error is small. Therefore, microfluidic chip technology has become a powerful tool for single cell analysis.
The multicellular system relies on intercellular interactions to coordinate cell signaling and regulate cell function, and understanding the mechanisms and processes of intercellular interactions is crucial for many physiological and pathological processes, such as embryogenesis, tissue regeneration, stem cell differentiation, cancer metastasis, etc. There is often a close interaction between different cell types, either homotypic or heterotypic, and depending on the distance between the cells, this can occur through a variety of mechanisms, such as: direct physical contact, diffusion of soluble secreted factors, electrical signaling, surrounding matrix of cells. In diseased tissues, for example, intercellular interactions can be disturbed, which has a crucial role in both the onset and progression of the disease. For another example, tumor sprouting is an important pathological feature of highly invasive behavior of tumors, and is closely related to the recurrence and metastasis of tumors. In the tumor microenvironment, the vascular endothelial cells migrate into the tumor under the action of factors such as VEGF secreted by the tumor cells, thereby providing conditions for the nutrient supply and invasion and metastasis of the tumor. The document Chung woosung; eum h.m.; lee h.o.; lee k.m.; leeh.b.; kim k.t.; ryu h.s.; kim s.; lee j.e.; park y.h. nature Communications,2017,8. 515 cells from 11 patients with breast cancer were collected and by single cell transcriptome analysis, the cancer cells showed common features in the early tumors and intratumoral heterogeneity in breast cancer subtypes and important pathways associated with cancer.
The development of methods for capturing, manipulating and analyzing single cells to study the heterogeneity of cells has important significance for studying cell growth, differentiation, disease diagnosis and monitoring. Although single cell capture, culture and analysis on a microfluidic chip is a good platform, the research on the separation and capture of two single cells of various cells and the interaction between single cells of various cells by using a microfluidic technology is not available at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a micro-fluidic chip and a design method and application thereof, the micro-fluidic chip has the advantages of miniaturized structure, low cost and high applicability, can realize the capture and culture of single cells and research the interaction between the two cells, and can reveal a certain intercellular interaction mechanism. The problem of separation and capture of two single cells in the background technology is solved, and the problem of research on interaction between single cells of various cells is also solved.
The technical scheme adopted by the invention for solving the technical problems is as follows: the microfluidic chip is formed by bonding a polydimethylsiloxane chip and a substrate carved with a channel, wherein the channel comprises a main channel, a capturing unit and a narrow channel;
the main channel is used for transmitting cells and cell culture media and comprises a first main channel and a second main channel which are arranged side by side; the side walls of the first main channel and the second main channel are oppositely arranged with a plurality of capture units at intervals, and the capture units are used for capturing and culturing single cells; the narrow channel is used for communicating the capture unit of the first main channel with the capture unit at the corresponding position of the second main channel, forming an alternating current channel between the captured single cells and preventing the captured single cells from flowing into the opposite main channel.
In a preferred embodiment of the invention, the width of the capture unit decreases from the main channel to the narrow channel, and the narrow channel corresponds to a valve when a cell enters the capture unit.
In a preferred embodiment of the present invention, the cross-sectional shape of the capturing unit is a semi-ellipse or a triangle. The invention adopts a method for the position relationship between the fluid streamline and the captured cells, and the position of the cells in the capture unit can be guided through the direction of the streamline, so that the condition of the next cell flow is influenced. Under the condition of not changing the length and the width of the main channel, the width and the length of the gap and the length and the width of the capture unit, the shape of the capture unit is changed, and the influence on the capture position of the cells is checked.
In a preferred embodiment of the present invention, the width of the main channel is 20 to 40 μm, the spacing distance between the capturing units on the same main channel is 100 to 200 μm, the entrance dimension of the capturing units is 20 to 35 μm, the width of the narrow channel is 2 to 10 μm, the length of the narrow channel is 5 to 10 μm, and the height of the channel is 20 to 40 μm.
In a preferred embodiment of the present invention, the width of the main channel is 30 μm, the spacing distance between the capture units on the same main channel is 150 μm, the entrance dimension of the capture units is 25 μm, the width of the narrow channel is 5 μm, the length of the narrow channel is 6 μm, and the height of the channel is 25 μm.
The invention also provides a design method of the microfluidic chip, which comprises the following steps:
1) using the "minimum path of flow resistance" principle, the flow of fluid in a channel typically occurs at a low Reynolds number (Re), and for a rectangular channel laminar flow without a drop and obstruction, the resistance is calculated as
Figure BDA0002199023870000031
Wherein μ is the viscosity of the fluid, Q is the flow rate, Q ═ V × a, V is the flow velocity, a is the cross-sectional area of the channel, L, w, h are the length, width and height of the channel, respectively, α is a dimensionless parameter related to the aspect ratio,
Figure BDA0002199023870000032
Figure BDA0002199023870000033
2) calculating the pressure drop Δ P:
for the resistance of the capture unit, approximately calculated as a rectangular resistance;
pressure drop of fluid through the first main channel and the narrow channel, wherein P0Denotes a narrow channel, P1Representing a first main channel
Figure BDA0002199023870000041
Pressure drop of fluid through the narrow channel and the second main channel, where P2Representing the second main channel
Figure BDA0002199023870000042
3) Because the height of the narrow channel is larger than the width, the height h and the width w of the formula are exchanged to obtain the following formula
Figure BDA0002199023870000043
4) When the pressure drop of the first main passage and the second main passage are the same,
Figure BDA0002199023870000044
Qcapture unit＝QNarrow channel ⑧；
Figure BDA0002199023870000045
5) Substituting numerical calculation, and designing the channel size according to the size of the cell to be captured.
The invention also provides application of the microfluidic chip in single cell capture.
In a preferred embodiment of the present invention, the application is single cell capture of two kinds of cells, the single cell capture of a first kind of cells is performed in a first main channel, and the single cell capture of a second kind of cells is performed in a second main channel after the captured cells are cultured and attached to the wall.
The invention adopts a method for simulating cell movement conditions by three-dimensional modeling, which comprises the following steps: and (3) constructing a cell model to simulate the flow of the cell model in the microchannel by using a particle tracking module in COMSOL software, and proposing that the captured cells on one side can be captured only after the captured cells on the other side are cultured to be attached to the walls. When the cell is suspended, the volume of the cell is equivalent to a micro valve, most of the fluid flows over the cell, and the height of the narrow channel is equivalent to the change
Figure BDA0002199023870000051
And after the cells captured at one side are cultured and attached to the wall, HEffective heightIs increased at this time
Figure BDA0002199023870000052
Cell capture on the other side can be achieved. And a more reliable basis is provided for the manufacture of the chip.
In a preferred embodiment of the present invention, the application is to study intercellular interaction, single cell capture is performed in both the first main channel and the second main channel, and cytokines produced by cells are mutually transmitted by diffusion, thereby realizing study of intercellular interaction.
In a preferred embodiment of the invention, a particle tracking module in COMSOL software is utilized to carry out three-dimensional modeling on a microfluidic chip to simulate the cell movement condition; or a dilute substance transfer module in COMSOL software is used for simulating the signal transfer condition between the cells captured by the microfluidic chip.
The microfluidic chip for single cell capture and study of intercellular interactions of the present invention exhibits some significant advantages, including: the preparation is simple, easy to operate, the used material has good biocompatibility, the cell culture condition is close to the microenvironment in vivo, higher single cell capture efficiency can be realized, the transmission of intercellular signals can be carried out, and a good device is provided for researching the interaction between cells. The micro-fluidic chip can be used for developing the research of tumor budding mechanism, providing basis and new research thought for constructing an in vitro effective tumor budding model, and providing certain theoretical support for the generation and development of tumors.
Drawings
FIG. 1 is a drawing of a microfluidic chip.
Fig. 2A shows the structure of the microfluidic chip, B shows the actual microfluidic chip, and C, D and E show the mixing of two dyes.
FIG. 3 is a schematic diagram of a single-cell capture principle and a simulation structure of the microfluidic chip.
FIG. 4 is a graph showing velocity profiles of (a)100 μm, (b)150 μm, (c)200 μm different main channel lengths, and (d) channel length ratio vs. Q.
Fig. 5 shows a velocity profile, a velocity flow chart, and a pressure profile of the capturing unit from top to bottom in the order of triangle (a), (b) semi-ellipse, (c) rectangle, (d) circle.
FIG. 6(a), (b), (c) channel entrance velocity optimization and (d) pressure relationship after primary cell capture, on the other side of the captured cells.
FIG. 7 optimizes the entrance width of the single-sided capture unit, (a) the entrance velocity 5 μm/s capture unit velocity flow diagram, (b) the particle tracking module in COMSOL simulates cell movement.
FIG. 8 is a graph of the velocity profile of the fluid on the other side of the three-dimensional simulation of the captured cell suspension, (a) the three-dimensional velocity profile of the narrow channel, and (b) the y-z sectional velocity profile of the narrow channel.
FIG. 9 is a three-dimensional simulation of the movement of particles on the other side when cells adhere to the wall, and (a) a three-dimensional velocity distribution diagram of a narrow channel and (b) a particle flow position distribution diagram.
FIG. 10 is a graph simulating diffusion of intercellular interacting substances.
Detailed Description
The invention is explained in detail below with reference to the drawings and examples:
example 1
Referring to fig. 1, the fabrication of the microfluidic chip of this embodiment adopts a soft lithography method, which includes the following steps:
manufacturing a mould: heating the silicon wafer in a water and acid solution until boiling for 30min, cleaning with ultrapure water, drying by blowing with nitrogen, throwing a layer of SU-82015 negative photoresist on the surface by a spin coater at the rotation speed of 2500rpm, baking for 10min at 65, baking for 4min at 95m, exposing for 3 times by an ultraviolet exposure machine for 30s each time, and drying by blowing with nitrogen after developing, namely obtaining a pattern captured by cells on the silicon wafer.
Manufacturing a chip: adding trifluoro (1H,1H,2H,2H perfluorooctyl) silane and a mould into a drying vessel, keeping the vacuum for 2 hours, then leading the surface of the mould to be hydrophobic, mixing Polydimethylsiloxane (PDMS) prepolymer and an initiator according to the mass ratio of 10:1, pouring the mixture into the mould, removing bubbles, and then placing the mixture for 2 hours at 75 ℃. And (3) removing the polymerized PDMS sheet, cutting and molding, punching by using a flat-head injector needle, and bonding the PDMS and the glass after plasma treatment, so that the chip is successfully manufactured.
Referring to fig. 2 and 3, the microfluidic chip is formed by bonding a polydimethylsiloxane chip and a substrate engraved with channels, wherein the channels comprise a main channel, a capture unit and a narrow channel;
the main channel is used for transmitting cells and cell culture media and comprises a first main channel and a second main channel which are arranged side by side; the two main channels are arranged on the chip in parallel or in a bent manner to form a capture array;
the side walls of the first main channel and the second main channel are oppositely arranged with a plurality of capture units at intervals, and the capture units are used for capturing and culturing single cells; the narrow channel is used for communicating the capture unit of the first main channel with the capture unit at the corresponding position of the second main channel, forming an alternating current channel between the captured single cells and preventing the captured single cells from flowing into the opposite main channel.
The width of the capture unit decreases from the main channel to the narrow channel. The width of the main channel is 30 micrometers, the spacing distance of the capture units on the same main channel is 150 micrometers, the inlet size of the capture units is 25 micrometers, the width of the narrow channel is 5 micrometers, the length of the narrow channel is 6 micrometers, and the height of the microfluidic chip is 25 micrometers.
Examples 2 to 4
The design method of the sizes of all parts of the microfluidic chip comprises the following steps:
1) using the "minimum path of flow resistance" principle, the flow of fluid in a channel typically occurs at a low Reynolds number (Re), and for a rectangular channel laminar flow without a drop and obstruction, the resistance is calculated as
Wherein μ is the viscosity of the fluid, Q is the flow rate, Q ═ V × a, V is the flow velocity, a is the cross-sectional area of the channel, L, w, h are the length, width and height of the channel, respectively, α is a dimensionless parameter related to the aspect ratio,
Figure BDA0002199023870000081
Figure BDA0002199023870000082
2) calculating the pressure drop Δ P:
for the resistance of the capture unit, approximately calculated as a rectangular resistance;
pressure drop of fluid through the first main channel and the narrow channel, wherein P0Denotes a narrow channel, P1Representing a first main channel
Figure BDA0002199023870000083
Pressure drop of fluid through the narrow channel and the second main channel, where P2Representing the second main channel
Figure BDA0002199023870000084
3) Because the height of the narrow channel is larger than the width, the height h and the width w of the formula are exchanged to obtain the following formula
Figure BDA0002199023870000085
4) When the pressure drop of the first main passage and the second main passage are the same,
Figure BDA0002199023870000086
Qcapture unit＝QNarrow channel ⑧；
Figure BDA0002199023870000087
5) Substituting numerical calculation, and designing the channel size according to the size of the cell to be captured.
Examples 2-4 examined three cases of 100 μm, 150 μm and 200 μm, respectively, and the fluid flow simulation was performed using COMSOL software;
the flow rate of the fluid at inlet 1 was set to 1 μ l.min for the inlet 1 and inlet 2 positions shown in fig. 3-1，V＝0.0267m.s-1，LInlet length, laminar flowAn inlet length that develops laminar flow. Water was used as the material for the simulation.
Figure BDA0002199023870000091
ρWater (W)＝1000kg/m3，μWater (W)＝1.01×10-3Pa·s，
Figure BDA0002199023870000092
The change in color within the channel indicates the magnitude of the velocity. The inlet 2 is set as the outlet and the pressure is 0. Results of COMSOL simulation if shown in FIG. 4, QNarrow channel/QMain channelIncreasing with increasing main channel length, when the main channel length is 150 μm, QNarrow channel/QMain channelGreater than 1, but the pressure inside the chip will increase, and the PDMS and the glass bonded chip can bearThe pressure is limited.
Examples 5 to 8
The length of the main channel between the capturing units is 150 μm, the width and length of the entrance of the capturing units are unchanged, the shapes of the capturing units are changed, and the shapes of the capturing units of examples 3-8 are triangular, semi-elliptical, square and circular.
As shown in FIG. 5, the four shapes of the capturing units have no influence on the flow resistance, but the fluid streamlines passing through the capturing units are different, and the fluid is more concentrated when the capturing units are triangular and semi-elliptical, so that the concentration of cells in the capturing units can be reduced. Cells occupy a larger area ratio. Whereas circular, rectangular shapes are more non-linear in the capture unit and the pressure diagram shows that circular, rectangular shapes are also more prone to shear forces at the junction of the main channel and the capture unit.
Respectively performing single cell capture analysis on the first cell and the second cell
The cell enters the capture unit, like a valve closing the inlet of the capture unit, and most of the fluid does not pass, resulting in QNarrow channel/QMain channelIs less than 1. The next cell enters a second capture unit, and so on, so that single-cell capture on one side is realized. In order to reduce the resistance of the second main channel liquid to enter the capture cell during capture in the second channel, the inlet of the first main channel was opened at a velocity of 0 μm/s and a pressure of 0 pa. Increasing the resistance to fluid flow in the second main channel due to the presence of the capture unit cells in the first main channel, by calculating QNarrow channel/QMain channel0.23-0.25.
As shown in fig. 6, it is not feasible to achieve single cell capture at the second main channel by increasing the entrance velocity of the second main channel. But also increases the pressure on the sides of the captured cells. The width of the cell flowing into the inlet of the capturing unit was increased under the conditions of constant flow rate, outlet pressure, and the like, as shown in FIG. 7, and the result Q wasgap/Qbypass< 1 and also cause non-linear accumulation of fluid. From the formula, Q is determinedgap/QbypassMainly comprisesThe length and width of the narrow channel region. The width of the entrance of the cell into the capture unit has little effect on the flow.
As shown in FIGS. 6 and 7, after one side of the cells are captured, the width of the inlet of the capturing unit cannot achieve Q by changing the speed of the inlet on the other sideNarrow channel/QMain channel(> 1), three-dimensional simulation was performed using COMSOL software. Stretching is performed by taking a two-dimensional graph as a working plane, the height of a channel is an important parameter, H must be larger than the diameter of a single cell, but H is too large, so that aggregation of multiple cells in a capture unit (phi < H < 1.4 phi) can realize one capture unit and one cell. Therefore, we set the height of the entire channel to 25 μm for the simulation. Based on the behavior of adherent cell flow, we set the cells captured by the first main channel to 1 μm from the bottom of the channel and set the cell diameter to 16 μm. Simulating the cell flow at the second main channel in a three-dimensional environment, setting the second main channel inlet velocity to 5 μm/s and the first main channel inlet velocity to 0 μm/s, the fluid in the narrow channel is concentrated at a position above the cell diameter, where most of the fluid flows, as shown in FIG. 8. When the cells are suspended, the volume of the cells is equivalent to a micro valve, most of the fluid flows over the cells, the height of a narrow channel is equivalent to change, and HEffective height＝HNarrow channel-RCells＜HNarrow channel，QNarrow channel/QMain channelAccording to the results of the two-dimensional simulation of fig. 6, changing the inlet velocity does not effectively improve the capturing efficiency, but instead increases the shear force applied to the cells on the other side, and the increase in velocity also causes the captured cells to be flushed back into the main channel.
The increase in the flow resistance of the capture unit is mainly the decrease in the effective height of the solution flow through the narrow channel after one side cell capture. Placing the unilaterally captured cells into CO2The culture was carried out in an incubator at 37 ℃ for a certain period of time. Live cells will grow adherent to the wall, reducing the height of the cells, and simulating the height of the cells after the cells are adherent to the wall to be 5 μm. As shown in FIG. 9, the effective height of narrow channel solution flow is increased compared to that of the suspension cells after adherent cell growth, where Q isNarrow channel/QMain channel(> 1), the flow through the narrow channels is much greater than the flow through the main channels. The cell capture can be realized, the morphology of dead cells is not changed, the speed of the inlet of the second main channel is properly increased, the dead cells can be flushed into the main channel again, and the cells captured at the side of the first main channel can be screened for death and survival.
Analysis of interaction between cells
And simulating the diffusion of substances among cells of the capture unit under the condition that the speeds of the inlet channels on the two sides are the same. As shown in fig. 10, intercellular substance diffusion can be performed efficiently, and substance exchange between cells is more sufficient as the channel length increases. Simulations found that the direction of cell concentration diffusion on both sides changed for every U-shaped channel passing through the bottom of the channel.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims and their equivalents.

Claims (10)

1. A micro-fluidic chip is formed by bonding a polydimethylsiloxane chip and a substrate carved with a channel, and is characterized in that: the channel comprises a main channel, a capture unit and a narrow channel;
the main channel is used for transmitting cells and cell culture media and comprises a first main channel and a second main channel which are arranged side by side; the side walls of the first main channel and the second main channel are oppositely arranged with a plurality of capture units at intervals, and the capture units are used for capturing and culturing single cells; the narrow channel is used for communicating the capture unit of the first main channel with the capture unit at the corresponding position of the second main channel, forming an alternating current channel between the captured single cells and preventing the captured single cells from flowing into the opposite main channel.
2. A microfluidic chip according to claim 1, wherein: the width of the capture unit decreases from the main channel to the narrow channel.
3. A microfluidic chip according to claim 2, wherein: the cross-sectional shape of the capturing unit is a semi-ellipse or a triangle.
4. A microfluidic chip according to claim 1, wherein: the width of the main channel is 20-40 mu m, the spacing distance of the capturing units on the same main channel is 100-200 mu m, the inlet size of the capturing units is 20-35 mu m, the width of the narrow channel is 2-10 mu m, the length of the narrow channel is 5-10 mu m, and the height of the channel is 20-40 mu m.
5. A microfluidic chip according to claim 1, wherein: the width of the main channel is 30 micrometers, the spacing distance of the capture units on the same main channel is 150 micrometers, the inlet size of the capture units is 25 micrometers, the width of the narrow channel is 5 micrometers, the length of the narrow channel is 6 micrometers, and the height of the channel is 25 micrometers.
6. The method for designing a microfluidic chip according to any one of claims 1 to 5, comprising the steps of:
1) using the "minimum path of flow resistance" principle, the flow of fluid in a channel typically occurs at a low Reynolds number (Re), and for a rectangular channel laminar flow without a drop and obstruction, the resistance is calculated as
Figure FDA0002199023860000011
Wherein μ is the viscosity of the fluid, Q is the flow rate, Q ═ V × a, V is the flow velocity, a is the cross-sectional area of the channel, L, w, h are the length, width and height of the channel, respectively, α is a dimensionless parameter related to the aspect ratio,
Figure FDA0002199023860000021
Figure FDA0002199023860000022
2) calculating the pressure drop Δ P:
for the resistance of the capture unit, approximately calculated as a rectangular resistance;
pressure drop of fluid through the first main channel and the narrow channel, wherein P0Denotes a narrow channel, P1Representing a first main channel
Figure FDA0002199023860000023
Pressure drop of fluid through the narrow channel and the second main channel, where P2Representing the second main channel
Figure FDA0002199023860000024
3) Because the height of the narrow channel is larger than the width, the height h and the width w of the formula are exchanged to obtain the following formula
Figure FDA0002199023860000025
4) When the pressure drop of the first main passage and the second main passage are the same,
Figure FDA0002199023860000026
Qcapture unit＝QNarrow channel ⑧；
5) Substituting numerical calculation, and designing the channel size according to the size of the cell to be captured.
7. The use of a microfluidic chip according to any of claims 1 to 5 for single cell capture.
8. Use according to claim 7, characterized in that: the application comprises single cell capture of two cells, the single cell capture of a first cell in a first main channel, and the single cell capture of a second cell in a second main channel after the captured cells are cultured and attached to the wall.
9. Use according to claim 7, characterized in that: the application is to research the interaction between cells, single cells are captured in the first main channel and the second main channel, and cytokines generated by the cells are mutually transmitted through diffusion, so that the research on the interaction between cells is realized.
10. Use according to claim 7, characterized in that: carrying out three-dimensional modeling on the micro-fluidic chip by using a particle tracking module in COMSOL software to simulate the cell motion condition; or a dilute substance transfer module in COMSOL software is used for simulating the signal transfer condition between the cells captured by the microfluidic chip.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112268934A (en) * 2020-09-16 2021-01-26 东南大学 Detection chip and detection method for circulating tumor cells
CN113088447A (en) * 2021-03-11 2021-07-09 温州医科大学 Suspended array microfluidic chip and preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112268934A (en) * 2020-09-16 2021-01-26 东南大学 Detection chip and detection method for circulating tumor cells
CN112268934B (en) * 2020-09-16 2022-06-28 东南大学 Detection chip and detection method for circulating tumor cells
CN113088447A (en) * 2021-03-11 2021-07-09 温州医科大学 Suspended array microfluidic chip and preparation method and application thereof

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